Technical Report on critical concentrations for drug susceptibility testing of isoniazid and the rifamycins (rifampicin, rifabutin and rifapentine)

The critical concentrations for culture-based phenotypic drug susceptibility testing (DST) to first-line anti-TB drugs have been revised by the the World Health Organization (WHO). Critical concentrations for rifampicin have been lowered while those for isoniazid have been maintained at the present level. This update helps address the discordance observed between phenotypic and molecular methods to detect rifampicin resistance and improves the accuracy of DST. As a result patients with TB will have a more accurate diagnosis. This document is the outcome of a Technical Expert Group meeting convened by WHO in 2020 to assess the results of a systematic review of published literature on critical concentrations for DST of the most important first-line anti-TB drugs, isoniazid and the rifamycins (rifampicin, rifabutin and rifapentine). These critical concentrations had not been revised since 2008. New evidence over the past decade showed that critical concentrations used for phenotypic methods to detect rifampicin resistance may incorrectly classify strains with certain mutations. The following media were considered: Löwenstein-Jensen (LJ), Middlebrook 7H10 (7H10), Middlebrook 7H11 (7H11) and BACTEC™ Mycobacterial Growth Indicator Tube™ 960 (MGIT). Guidance has been provided to resolve discordance between genotypic and phenotypic results for these drugs and areas for further research have been highlighted. DST methods continue to have a very important role to identify resistance not detected by molecular assays and to support the interpretation of molecular assays results. However, they require sophisticated laboratory infrastructure, qualified staff and strict quality assurance procedures.Peer reviewe

Updating the approaches to define susceptibility and resistance to anti-tuberculosis agents: implications for diagnosis and treatment

11 páginas, 2 figuras, 1 tablaInappropriately high breakpoints have resulted in systematic false-susceptible AST results to anti-TB drugs. MIC, PK/PD and clinical outcome data should be combined when setting breakpoints to minimise the emergence and spread of antimicrobial resistance.I. Comas was supported by PID2019-104477RB-I00 from the Spanish Science Ministry and by ERC (CoG 101001038)Peer reviewe

Clinical outcomes and safety of colistin in treatment of gram negative infections: A prospective observational study

Background: Despite the fact that colistin has a significant activity against MDR gram-negative organisms, its toxicity limits its use. However, the limited therapeutic options due to increasing antibiotic resistance have made re-evaluation of older antibiotics inevitable. In contrast, lack of data to guide the usage of these drugs demands for studies on their safety and efficacy. This studies the clinical outcomes and safety of colistin at a tertiary care centre in Mumbai. Materials and methods: A prospective observational study was conducted at P.D. Hinduja Hospital, Mumbai for a period of seven months. Diagnosis of infection was based on CDC guidelines and APACHE II score was used to assess the severity of illness. Clinical and microbiological response to colistin was evaluated along with the incidence of nephrotoxicity (RIFLE criteria) and neurotoxicity. Results: Sixty-two patients (median age 56 years, with documented gram negative bacterial infection and mean APACHE II score 22) received colistin. Clinically favourable response was seen in 71% patients. However, the mortality among the study population was 27%. Univariate analysis identified pneumonia and ICU admission as independent factors for adverse outcome. Deterioration of renal function was observed in 35.89% as per RIFLE criteria. 6 (9.6%) patients demonstrated neurotoxicity. Conclusion: Colistin is effective in treatment of gram negative infections and its use should be reappraised. However since colistin is the last resort it is imperative to make its best use to ensure that it remains as a safe and effective mode of treatment when need be

Pyrosequencing to resolve discrepant Xpert MTB/RIF and Mycobacterial Growth Indicator Tube 960

Delayed diagnosis of drug resistance has been a major obstacle to proper management and control of drug-resistant tuberculosis (TB). Expanded access to rapid molecular diagnostics such as Xpert MTB/RIF has been helpful, but has generated confusion about how to interpret genotype–phenotype discordance. Optimal management is not clearly defined for patients with rifampin resistance by Xpert MTB/RIF but rifampin susceptibility by phenotypic testing. To resolve this discrepancy, we performed pyrosequencing of discordant isolates identified at a reference laboratory over a 6-month period. We present here strategies to address genotype–phenotype discordance using sequencing

Evaluation of genotype MTBDRsl assay to detect drug resistance associated with fluoroquinolones, aminoglycosides and ethambutol on clinical sediments.

BACKGROUND: The emergence of resistant tuberculosis (TB) is a major setback to the global control of the disease as the treatment of such resistance is complex and expensive. Use of direct detection of mutations by molecular methods could facilitate rapid diagnosis of resistance to offset diagnostic delays. We evaluated the performance of the Genotype MTBDRsl (Hain Life Sciences) for the detection of second line resistant TB directly from stored smear positive sputum sediments. METHODOLOGY/PRINCIPAL FINDINGS: The assay showed a diverse range of sensitivity and specificity, 91.26% [95% CI, 84-96] and 95.5% [95% CI, 87-99] for FQ (PPV ∼97% & NPV ∼ 87.67%), 56.19% [95%CI, 46-66] and 81% [95%CI, 66-91] for EMB (PPV ∼ 88.06% & NPV ∼ 43.21%) and 100% for SLD. Diagnostic accuracy for FQ, SLD and EMB was 94%, 100% and 63.51%, respectively. 1.17% (2/170) were heteroresistance strains, where the heteroresistance was linked to rrs gene. A varying rate of validity was observed 100% (170/170) for FQ, 94.11% (160/170) for EMB, 88.23% (150/170) for SLD. CONCLUSIONS/SIGNIFICANCE: Genotype MTBDRsl is simple, rapid, economical assay that can be used to detect commonly known resistance associated with Fluoroquinolone, second line injectable drugs and ethambutol. The assay detects the targeted resistance in less time as compared to phenotypic DST. But due to low NPV to FQ (88%) and EMB (43.21%), the assay results must be interpreted in coordination with the phenotypic DST

Updating the approaches to define susceptibility and resistance to anti-tuberculosis agents: implications for diagnosis and treatment

[No Abstract Available]As current or former employees or consultants for FIND, the work of R.B. Baldan, I. Comas, C.M. Denkinger, D.L. Dolinger, S.B. Georghiou, C.U. Koser and T.C. Rodwell on the systematic reviews, including this viewpoint, was supported by Unitaid (grant 2019-32-FIND MDR), BMGF (grant OPP1105925), the German Federal Ministry of Education and Research through KfW, the Dutch Ministry of Foreign Affairs, the Australian Department of Foreign Affairs and Trade, and UK aid from the British people. N. Alvarez and J. Robledo are funded by MinCiencias, Colombia (number 221389622138966621666216 CT-783-2018). A. Aubry and N. Veziris work at the Centre National de Reference des Mycobacteries, which receives an annual grant from Sante Publique France and have received research grants from Janssen for studies on bedaquiline. P. Claxton and I.F. Laurenson are funded through National Services Scotland. I. Comas was supported by PID2019-104477RB-I00 from the Spanish Science Ministry and by ERC (CoG 101001038). M. Egger is supported by the Swiss National Science Foundation (grant number 320030_153442 and 189498) and the US National Institutes of Health, National Institute of Allergy and Infectious Diseases, the Eunice Kennedy Shriver National Institute of Child Health and Human Development, the National Cancer Institute, the National Institute of Mental Health, the National Institute on Drug Abuse, the National Heart, Lung, and Blood Institute, the National Institute on Alcohol Abuse and Alcoholism, the National Institute of Diabetes and Digestive and Kidney Diseases, the Fogarty International Center, and the National Library of Medicine: Asia-Pacific, U01AI069907; CCASAnet, U01AI069923; Central Africa, U01AI096299; East Africa, U01AI069911; NA-ACCORD, U01AI069918; Southern Africa, U01AI069924; West Africa, U01AI069919. M.R. Farhat is supported by NIH NIAID R01AI155765. S.K. Heysell was funded by NIH NIAID grants R01 AI137080 and U01 AI150508. T. Jagielski was supported by a DAINA grant (number 2017/27/L/NZ6/03279) from the National Science Centre, Poland. J.L. Johnson was supported by contracts NO1-AI95383 and NO1-AI-70022 of the US National Institutes of Health. P.M. Keller was supported by Innosuisse 36198.1 IP-LS. C.U. Koser is a research associate at Wolfson College and visiting scientist at the Department of Genetics, University of Cambridge. The Federal Government of Germany supported C.U. Koser as part of his work for the European Laboratory Initiative, WHO Regional Office for Europe. C.U. Koser was further supported by the Royal Society of Tropical Medicine and Hygiene and the National Institute for Health Research Cambridge Biomedical Research Centre and received an observership by the European Society of Clinical Microbiology and Infectious Diseases to the EUCAST Development Laboratory for Bacteria (Vaxjo, Sweden), hosted by Gunnar Kahlmeter and Erika Matuschek. D. Machado and M. Viveiros are funded in part by Fundacao para a Ciencia e a Tecnologia, Portugal (PTDC/BIA-MIC/30692/2017, UID/Multi/04413/2020 and DL57/CEECIND/0256/2017). S. Niemann is supported by the German Center for Infection Research, Excellenz Cluster Precision Medicine in Chronic Inflammation EXC 2167, Leibniz Science Campus Evolutionary Medicine of the LUNG (EvoLUNG). S.V. Omar has received funding to prepare and provide training for Janssen Pharmaceutica activities. L. Rigouts is supported by the Belgian Directorate General for Development. T.C. Rodwell was additionally funded in part by FIND and NIH NIAD, grants: P30 AI036214 and R21 AI135756. T.; Schon is funded by the Swedish Heart and Lung Foundation and the Swedish Research Council. T.R. Sterling has received funding from the US National Institutes of Health and the Centers for Disease Control and Prevention. G. Theron and R. Warren are supported by baseline funding from the South African Medical Research Council. R.J. Wilkinson receives funding from the Wellcome Trust (203135) and from the Francis Crick Institute, which is supported by Cancer Research UK (FC0010218), UKRI (FC0010218) and the Wellcome Trust (FC0010218). The views expressed here are those of the authors and do not necessarily correspond to those of their respective employers.Unitaid [2019-32-FIND MDR]; BMGF [OPP1105925]; German Federal Ministry of Education and Research through KfW; Dutch Ministry of Foreign Affairs; Australian Department of Foreign Affairs and Trade; MinCiencias, Colombia [221389622138966621666216 CT-783-2018]; Sante Publique France; Janssen; National Services Scotland; Spanish Science Ministry [PID2019-104477RB-I00]; ERC [CoG 101001038]; Swiss National Science Foundation [320030_153442, 189498]; US National Institutes of Health; National Institute of Allergy and Infectious Diseases; Eunice Kennedy Shriver National Institute of Child Health and Human Development; National Cancer Institute; National Institute of Mental Health; National Institute on Drug Abuse; National Heart, Lung, and Blood Institute; National Institute on Alcohol Abuse and Alcoholism; National Institute of Diabetes and Digestive and Kidney Diseases; Fogarty International Center; NIH NIAID [R01AI155765, R01 AI137080, U01 AI150508]; DAINA grant from the National Science Centre, Poland [2017/27/L/NZ6/03279]; US National Institutes of Health [NO1-AI95383, NO1-AI-70022]; Innosuisse [36198.1 IP-LS]; Federal Government of Germany; Royal Society of Tropical Medicine and Hygiene; National Institute for Health Research Cambridge Biomedical Research Centre; Fundacao para a Ciencia e a Tecnologia, Portugal [PTDC/BIA-MIC/30692/2017, UID/Multi/04413/2020, DL57/CEECIND/0256/2017]; German Center for Infection Research, Excellenz Cluster Precision Medicine in Chronic Inflammation, Leibniz Science Campus Evolutionary Medicine of the LUNG (EvoLUNG) [EXC 2167]; Belgian Directorate General for Development; FIND; NIH NIAD [P30 AI036214, R21 AI135756]; Swedish Heart and Lung Foundation; Swedish Research Council; Centers for Disease Control and Prevention; South African Medical Research Council; Wellcome Trust [FC0010218, 203135]; Francis Crick Institute - Cancer Research UK [FC0010218]; UKRI [FC0010218]; National Library of Medicine: Asia-Pacific [U01AI069907]; National Library of Medicine: CCASAnet [U01AI069923]; National Library of Medicine: Central Africa [U01AI096299]; National Library of Medicine: East Africa [U01AI069911]; National Library of Medicine: NA-ACCORD [U01AI069918]; National Library of Medicine: Southern Africa [U01AI069924]; National Library of Medicine: West Africa [U01AI069919

Correlating rrs and eis promoter mutations in clinical isolates of Mycobacterium tuberculosis with phenotypic susceptibility levels to the second-line injectables

Objective/background: The in vitro drug-susceptibility testing of Mycobacterium tuberculosis reports isolates as resistant or susceptible on the basis of single critical concentrations. It is evident that drug resistance in M. tuberculosis is quite heterogeneous, and involves low level, moderate level, and high level of drug-resistant phenotypes. Thus, the aim of our study was to correlate rrs (X52917) and eis (AF144099) promoter mutations, found in M. tuberculosis isolates, with corresponding minimum inhibitory concentrations of amikacin, kanamycin, and capreomycin. Methods: Ninety M. tuberculosis clinical isolates were analyzed in this study. The minimum inhibitory concentrations were determined by MGIT 960 for 59 isolates with resistance-associated mutations in the rrs and eis promoter gene regions, and 31 isolates with wild-type sequences, as determined by the GenoType MTBDRsl (version 1) assay. Results: The rrs A1401G mutation was identified in 48 isolates resistant to the second-line injectables. The eis promoter mutations C-14T (n=3), G-10C (n=3), G-10A (n=3), and C-12T (n=2) were found within 11 isolates with various resistance profiles to the second-line injectables. Thirty-one isolates had wild-type sequences for the rrs and eis promoter gene regions of interest, one of which was amikacin, kanamycin, and capreomycin resistant. The isolates with the rrs A1401G mutation had amikacin, kanamycin, and capreomycin minimum inhibitory concentrations of >40 mg/L, >20 mg/L, and 5–15 mg/L, respectively. The isolates with eis promoter mutations had amikacin, kanamycin, and capreomycin minimum inhibitory concentrations of 0.25–1.0 mg/L, 0.625–10 mg/L, and 0.625–2.5 mg/L, respectively. Conclusion: This study provides a preliminary basis for the prediction of phenotypic-resistance levels to the second-line injectables based upon the presence of genetic mutations associated with amikacin, kanamycin, and capreomycin resistance. The results suggest that isolates with eis promoter mutations have consistently lower resistance levels to amikacin, kanamycin, and capreomycin than isolates with the rrs A1401G mutation